Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.


INTRODUCTION
Brain cancer remains the leading cause of cancer-related death in children [1].
Novel therapies with specific anti-tumor efficacy and less off-target toxicity hold promise to improve both survival rates and quality of life for MB patients.
OLIG2 is a basic helix-loop-helix (bHLH) transcription factor that functions in the developing brain in a bivalent manner, promoting differentiation in the oligodendrocyte lineage while maintaining neural stem and progenitor cells in an undifferentiated state [11][12][13]. OLIG2 phosphorylation plays a critical role in determining this bivalent function [14,15]. In brain tumors, the anti-differentiation function of OLIG2 plays an important role in tumor progression, as seen in glioblastoma (GBM) [16] and in SHH MB [17]. In SHH MB, moreover, OLIG2-expressing stem cells promote recurrence after cytotoxic chemotherapy and radio-resistance [18]. These data support a model in which MB stem cells within this heterogeneous tumor microenvironment are inadequately treated by standard therapy and drive recurrence.
Considering the role of OLIG2 in restricting neural stem cell differentiation, and the role of OLIG2-expressing tumor stem cells in SHH-MB recurrence, we investigated whether pharmacologic disruption of OLIG2 would enhance MB therapy by targeting stem cells that may be resistant to radiation and cytotoxic chemotherapy. As OLIG2+ cells make up only a fraction of the proliferative population within MB, we expected that OLIG2 inhibitor therapy would require combination with additional therapies for optimal anti-tumor effect. We therefore investigated the efficacy of anti-OLIG2 therapy in both single agent format, to define potential anti-tumor effects, and in multi-modal combinations as are typically needed in clinical treatment of MB patients.
OLIG2, like other bHLH transcription factors, requires dimerization to initiate function [19]. Therefore, this dimerization interface presents an opportunity for targeted disruption [20]. A series of small molecule OLIG2 inhibitors were previously identified using a pharmacophore-guided 3D-structural search to find compounds that engage the OLIG2 dimerization interface [21]. The ability of these compounds to disrupt OLIG2 dimerization was demonstrated and quantitatively measured using fluorescence cross-correlation spectroscopy (FCCS), which measures the temporal correlation between signals from OLIG2 molecules individually tagged with either eGFP or Tomato, after co-transfection [22]. These methods showed that the disruptive effects of different agents on OLIG2 dimerization correlated with the inhibitory effects of these agents on in vitro tumor cell growth [21,22], supporting the proposed mechanism of action of these agents and the specificity of their anti-tumor effect.
In this study we analyzed OLIG2 expression in MB subgroups and evaluated the effect of OLIG2 inhibition on tumor growth, either as a single intervention and in combinatorial therapies. We investigated OLIG2 inhibition using CT-179, a compound based on the previously studied agent SKOG102. CT-179 is a novel small molecule OLIG2 inhibitor developed by Curtana Pharmaceuticals (molecular weight (MW) 397.3 g/mol, patent application WO2016138479A1) that has shown favorable blood brain barrier (BBB) penetration and stability in preliminary animal studies. CT-179 was recently awarded FDA Rare Pediatric Disease Designation for the treatment of MB.
We evaluated CT-179 using MB cell lines, explant tissue organoids (MBOs) and in vivo models that are prone to recurrence, including patient-derived xenografts (PDX) and a genetically-engineered mouse (GEM) model, analyzed using FCCS, kinomic analysis, in vitro and in vivo cell cycle analysis, in vivo tumor treatment studies and scRNA-seq. Our findings validate the potential of targeting OLIG2 in SHH-MB and highlight the novel brain penetrant small molecule CT-179 for further clinical evaluation, particularly when combined with radiotherapy (RT) in patients with MB.

OLIG2 correlates with poor survival in SHH-MB patients
Analysis of OLIG2 expression supported a significant role in SHH-MB heterogeneity.
Previous studies have shown that OLIG2 significantly correlated with patient outcome in SHH-MB patients [17]. To investigate the relationship of OLIG2 to survival in SHH-MB in specific subtypes, we analyzed transcriptomic datasets previously published by Cavalli and colleagues [23]. Results show that OLIG2 expression correlated with for poor patient outcomes in alpha, delta and gamma subtypes of SHH-MB, reaching significance for the alpha and gamma patient cohorts ( Figure 1A, Supplementary Figure 1A). Next, we analysed OLIG2 mRNA expression in a panel of immortalized MB lines, primary MB lines and PDX models [24]. OLIG2+ cell lines showed more abundant OLIG2 mRNA compared to OLIG2+ PDX tumors, consistent with heterogeneous expression in the tumors ( Figure 1B, Supplementary Figure 1B).
Western blots confirmed translation of OLIG2 mRNA in MB PDX and cell lines ( Figure   1C). In addition, immunohistochemistry (IHC) staining for OLIG2, performed on MB xenograft tumors, showed positive focal OLIG2 staining ( Figure 1D, Supplementary Figure 1C). Taken together, these findings highlight a potential role for OLIG2 in both inter-and intra-tumoral heterogeneity.

OLIG2 down-regulation induces G2/M phase cell cycle arrest and apoptosis
For initial analysis of OLIG2 function in MB, we silenced OLIG2 using small interfering  Figure 1D) and protein level ( Figure 1E).
Low-level OLIG2 protein expression persisted in cells treated with sequence #3 but OLIG2 was undetectable in cells treated with sequence #1 and #2. The degree of OLIG2 silencing correlated with the observed apoptotic response with cell death observed with sequence #1 and #2 but not sequence #3 ( Figure 1F).
Prior studies show OLIG2-dependent regulation of multiple cell cycle-associated transcriptional regulators and genes controlling microtubule function during development [25,26]. We therefore assessed the cell cycle following OLIG2 KD.
OLIG2 KD disrupted cell cycle progression, resulting in decreased cyclin dependent kinase 1 (CDK1) and phosphorylated CDK1 (p-CDK1) and increased expression of Mphase specific marker phosphorylated histone H3 (p-HH3) ( Figure 1G). Consistent with these changes, down-regulation of OLIG2 led to an increased proportion of cells in G2/M phase ( Figure 1H). OLIG2 KD also altered nuclear morphology, demonstrated by Hoescht (cyan) and altered alignment, demonstrated by ⍺-tubulin immunofluorescence (IF) (yellow). In contrast, after OLIG2 KD, cells showed a multinucleated/tetraploid appearance ( Figure 1I). Cell proliferation and apoptosis were assessed following KD using real-time live cell imaging; representative images are shown in Figure 1J and Supplementary Figure 1E. Quantification showed significant anti-proliferative and apoptotic effects using sequence #1 and #2 but less so using the less effective sequence #3 ( Figure 1K). These data demonstrate an important role of OLIG2 in the regulation of MB cell cycle progression and apoptosis, suggesting the therapeutic potential of targeting OLIG2.

CT-179 disrupts OLIG2 function by interfering with dimerization.
To investigate the therapeutic potential of pharmacologically disrupting OLIG2, we employed CT-179, a small molecule based on the previously described OLIG2 inhibitor SKOG102 [21,22]. FCCS uses confocal microscopy to quantify interactions between fluorescently tagged molecules in a minute observation volume, in which correlation between different fluorescent signals varies directly with their physical interaction (Supplementary Figure 2A). Prior FCCS studies in live cells showed that SKOG102 disrupts OLIG2 dimerization and DNA binding. We therefore used FCCS similarly to analyze the effect of CT-179 on OLIG2 dimerization using live HEK293 cells co-transfected with OLIG2-eGFP and OLIG2-Tomato fusion constructs. Confocal laser scanning microscopy (CLSM) showed that both fluorescently tagged OLIG2 proteins were localized in the cell nucleus in untreated cells, and that treatment with CT-179 did not change OLIG2 intracellular localization (Supplementary Figure 2B). In addition to these analyses, FCCS provide information about diffusion of OLIG2-eGFP in and out of the observation volume, based on the decay time of the autocorrelation curves. Fitting the data using a 2-component pure diffusion model, two diffusion times could be identified, designated as free OLIG2 diffusion (τD1), and OLIG2 diffusion slowed down by its binding to the DNA (τD2), which was markedly longer (τD2 >> τD1). Average diffusion time of free OLIG2-eGFP was determined to be τD1 = 700 μs, which was close to the value of free OLIG2-eGFP diffusion in the cell nucleus that was These results provide evidence that CT-179 reduced OLIG2-DNA binding, as expected to result from a reduction of OLIG2 dimerization.
To determine if CT-179 interfered with OLIG2 transcriptional activation, we developed a luciferase reporter assay, using Daoy cells to provide a relevant cellular context.
Prior ChIP-Seq studies showed that OLIG2 directly interacts with the promoter for LHX8, a gene that regulates neuronal differentiation [27]. We transfected Daoy cells with a luciferase reporter fused to the promoter region of the human LHX8 gene on then measured luciferase expression. Co-transfection with an OLIG2 expression construct induced strong luciferase expression, validating the LHX8 promoter as a reporter of OLIG2-dependent transcription ( Figure 2E). CT-179 treatment significantly decreased luciferase expression, demonstrating that CT-179 specifically blocked OLIG2-driven transcription.

CT-179 shows minimal off-target effects
To detect un-intended inhibitory effects on diverse processes, we used kinome profiling. Testing the effect of 1 µM CT-179 on more than 400 kinases identified 3 kinases that showed potentially significant inhibition, Fetal Liver Tyrosine Kinase 3 (FLT3), Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2) and KIT. DDR2 showed 80% inhibition, FLT3 showed 84% inhibition and KIT showed 66% inhibition ( Figure 2F, Supplementary Data 1, Kinome Scan). These kinases were selected for a dose response follow-up assay. The most potently inhibited kinase was FLT3 with an IC50 of 20 nM under these assay conditions. However, accounting for intracellular substrate availability using the Cheng-Prusoff equation and assuming 1 mM ATP, the cell potency of CT-179 to inhibit FLT3 is predicted to be IC50 > 3.4 µM [28]. We considered this potential inhibition to be negligible because free drug concentrations in vivo would be approximately 10 -100 times lower than the predicted IC50. These data indicate that CT-179 does not show any kinase inhibitory effect that would be relevant in vivo.
We analyzed the potential for CT-179 off-target toxicity using the BioMAP ® Diversity PLUS ® platform, a cell-based screen used to assess the safety of pre-clinical Importantly no cytotoxicity was observed even at 2.5 µM. Thus, in kinomic and cytotoxicity assays, CT-179 showed minimal off-target effects.

In vivo pharmacokinetic studies of CT-179
Two independent CT-179 pharmacokinetic (PK) animal studies, including brain PK, were performed by Biodura Inc. The initial study assessed the bioavailability and concentration versus time of CT-179 in mice administered either a single oral dose (per os, PO) (20 mg/kg) or a single intravenous (IV) dose (1 mg/kg) ( Figure 2H). Three mice (C57Bl/J6) were tested in each of the IV and PO groups. Results highlighted consistent plasma concentrations, which were maintained across a 24-hour period, indicating a suitable half-life for once per day dosing. Increasing CNS exposure was detected 4 hours after the initial PO dose, with an estimated brain to plasma (B/P) ratio reaching an average of 6.72 ( Figure 2I). The objective of the second study was to estimate steady state exposures of CT-179 in mice receiving PO doses of 1 and 5 mg/Kg ( Figure 2J). Results show both PO doses achieved significant plasma levels.
CT-179 exposures were measured in the plasma and brain at 24 hours on Days 1 and 3 to compare the initial dose to steady state levels (three doses) (Supplementary Data 1, PK). Findings indicate that CT-179 levels increased approximately 35% between the two time points at the 5 mg/kg PO dose (Supplementary Data 1, PK). CT-179 displayed an estimated brain to plasma ratio of >10, demonstrating CNS penetration and accumulation. While the B/P ratio was well above one, once a steady-state plasma concentration was achieved following repeat dosing, an equilibrium was achieved and the brain tissue concentration did not continue to rise with prolonged exposure. When CT-179 was discontinued, the drug concentration in the brain decreased over time (Supplementary Data 1, PK, 48 hours after Day 3 timepoint). CT-179 displayed a predicted half-life in the range of 10-12 hours and high bioavailability (range estimated as 58-91%; mean 75%) ((Supplementary Data 1, PK). Taken together, the PK studies demonstrated that CT-179 had high oral bioavailability and achieved measurable plasma and brain exposures over 24 hours with somewhat increased exposure between Days 1 and 3 following once daily dosing.

CT-179 induces apoptosis and mitotic arrest
Considering that our FCCS studies of CT-179 confirmed the predicted mechanism of OLIG2 inhibition and that CT-179 showed low off-target effects and the favorable PK, we analyzed the anti-tumor potential of CT-179 in live cells in culture. We selected Reduced expression of cleaved poly-ADP ribose polymerase (PARP) coincided with increasing cleaved caspase-3 overtime, indicating a robust apoptotic response ( Figure   3E). The apoptotic response to CT-179 correlated with reduced abundance of antiapoptotic proteins MCL-1, BCL-2, and BCL-xL ( Figure 3F). We also compared the effects on the cell cycle following CT-179 (1 µM) treatment alone versus in combination with RT (2 Gy). Results show a G2/M phase arrest following single therapy, which was more pronounced when combined with RT ( Figure 3G). Molecular analysis showed that CT-179 disrupted key mitotic mechanisms. Mitotic cells typically express cyclin B1 at the onset of mitosis when bound CDK1 mediates spindle assembly and mitotic entry [29], then rapidly degrade cyclin B1 after the spindle assembly checkpoint. Simultaneously, cells typically inactivate CDK1 by dephosphorylation and phosphorylate Histone H3 which resolves following telophase [29]. In Daoy cells, CT-179 treatment decreased cyclin B1, CDK1 and p-CDK1, while maintaining persistently elevated p-HH3 (Supplementary Figure 3C). Med-813 responded to CT-179 with a more complex disruption of mitotic mechanisms, with initial increase in cyclin B1, CDK1 and p-HH3 over 24 hours with decreased p-CDK1, followed by a decrease in cyclin B1 and CDK1 and a marked increase in p-CDK1 at later time points ( Figure 3H). To explore the effects of CT-179 on nuclear morphology and mitotic spindle formation, Hoechst (cyan) staining of the nucleus and α-tubulin (yellow) staining of mitotic spindles was conducted. IF staining results illustrated cells treated with vehicle had a normal nuclear morphology and spindle alignment, whereas cells treated with CT-179 took on a multinucleated/tetraploid appearance, with satellite micronuclei (white arrow) and an ancillary nuclear lobe formation (green arrow) ( Figure   3I). These studies show that CT-179 treatment delayed growth, induced cell killing and resulted in profound mitotic disruption.

CT-179 treatment induces cell death in MB explant organoids (MBOs)
MB in patients show cellular heterogeneity, and as CT-179 specifically targets the OLIG2-expressing fraction of cells, it is important to evaluate CT-179 in MB models that preserve tissue heterogeneity. For this purpose, we generated explant organoids directly from tumors surgically resected from patients without cell dissociation. This approach allows tumor stroma, blood vessels and immune infiltrate to remain intact.
We adapted a recent adult GBM explant approach [30, 31] to freshly resected MB specimens. To the best of our knowledge, we are the first group to adopt this technique to medulloblastoma, generating MBOs that could be cultured for up to 12 weeks. These studies demonstrated that combination therapy significantly reduced proliferation without inducing a compensatory increase in the SOX2+ population ( Figure 4C).

CT-179 shows additive anti-tumor effects with radiotherapy in vivo
To determine if CT-179 showed similar efficacy in vivo against human MB, we studied CT-179 both as a single agent or combined with RT in immune-compromised NOD  Figure 5E). These toxicities reflect that RT and CT-179 were administered at close to the maximum tolerated dose, and were not incompatible with clinically tolerable brain tumor therapy.

CT-179 disrupts OLIG2 processing and cell cycle progression in SHH-driven tumors
In an alternative approach to xenograft models that would enable in vivo studies of CT-179 in models with typical MB heterogeneity and tumor microenvironment (TME), We administered CT-179 via intraperitoneal (IP) injection, testing a range of doses from, 50-150 mg/kg. We found that the maximum tolerated dose (MTD) was 80 mg/kg EOD or 100 mg/kg every three days. Doses above this MTD were associated with brain hemorrhage and early animal deaths (Supplementary Figure 6A,B). In contrast, no hemorrhage was observed at 80 mg/kg EOD or 100 mg/kg every three days.
As an initial test of effect, we administered CT-179 (80 mg/kg) at P10, P12, P14 and P16 and then analyzed tumors by western blot 24 hours after the last dose, comparing phosphorylated OLIG2 (p-OLIG2) as marker of OLIG2 processing and phosphorylated RB (p-RB) as a marker of proliferation. CT-179-treated tumors showed decreased p-OLIG2, consistent with disruption of OLIG2 dimerization, and reduced p-RB, indicating an overall decrease in proliferation ( Figure 5A). Reduced p-OLIG2 supports the on-target specificity of CT-179 in vivo and reduced p-RB shows that disrupting the OLIG2+ subpopulation produced a detectable anti-tumor effect.
We measured the effect on tumor cell cycle dynamics in vivo at 6 and 24 hours after a single 80 mg/kg dose of CT-179. To label cells at S-phase, animals were injected with EdU at 40 mg/kg via IP 30 minutes prior to harvest. We dissociated tumors,

CT-179 reduces tumor growth in G-Smo mice with SHH-MB
To assess the in vivo efficacy of CT-179 longitudinally, we crossed Gli-luc mice that showed that the fraction of OLIG2 + cells increased in mice treated with CT-179 from P10-P17 ( Figure 5I). In the context of reduced p-OLIG2, the increase in total OLIG2 expression suggests a homeostatic response to OLIG2 inhibition that may contribute to the observed resistance in single-agent treatment.  Figure 5J). We noted potential for combinatorial toxicity, as initial studies combining CT-179 80 mg/kg EOD with 5 fractions of 2 Gy RT resulted in shortened survival times, and we accordingly adjusted the radiation dose downward (Supplementary Data 1, Regimens). We found that CT-179 plus 3 fractions of 0.5 Gy were tolerable and produced a statistically significant increase in EFS compared to either treatment alone, or to untreated controls ( Figure 5J). The addition of RT thus increased CT-179 efficacy, and conversely CT-179 sensitized tumors to RT in a model of highly aggressive and treatment-refractory SHH-MB.

CT-179 treatment alters MB cellular heterogeneity
As CT-179 specifically targets the OLIG2+ subset of tumor cells in MB, we used scRNA-seq to identify changes in cellular heterogeneity during CT-179 treatment. We CT-179 altered specific stromal populations but did not deplete either of the two oligodendrocyte clusters (Table 1). Mature, myelinating oligodendrocytes showed no significant change while immature oligodendrocytes increased ( Table 1)

Proliferative cells in CT-179-treated MBs up-regulate CDK4
To identify genes that promote proliferation during with CT-179 therapy, we compared gene expression in the proliferative clusters ( Figure  PA2G4 as the transcription factor with the highest relevance to this set 106 differentially up-regulated genes, with 23 included in both the differential gene set and the set of putative PA2G4 targets determined by ChEA3. Importantly, we did not observe a pattern of gene expression changes consistent with p53-mediated transcriptional regulation, indicating that a DNA damage response was not initiated. Consistent with PA2G4-mediated transcriptional regulation driving the differential gene expression pattern in proliferating cells in CT-179 treated tumors, the Pa2g4 transcript was within the set differentially up-regulated genes, and was up-regulated in each of the individual CT-179-treated replicate tumors compared to controls ( Figure   6I). Moreover, PA2G4 is associated with CDK/RB/E2F signalling [42,43], and CDK4, another element in the CDK/RB/E2F axis was similarly significantly enriched in cells that remained proliferative in CT-179-treated mice (Table S5). Like Pa2g4, Cdk4 was up-regulated in each CT-179-treated replicate ( Figure 6I). Considering the potential for CDK4 to act upstream of PA2G4 via RB/E2F signaling, and the availability of small molecule inhibitors of CDK4 that have been tested in mouse MB models [44][45][46], we selected CDK4 for further study as a candidate resistance mechanism.

Palbociclib and CT-179 are mutually enhancing in vivo
We investigated whether CDK4 up-regulation enabled MB growth during CT-179 treatment using a Pox-Palbo, a polyoxazoline nanoparticle formulation of the CDK4/6 inhibitor palbociclib. We previously showed that POx-Palbo suppresses CDK4/6 activity and MB growth more effectively than conventional palbociclib. Similar to CT-179, POx-Palbo produced an anti-tumor effect that was limited by recurrence. We examined whether blocking CDK4 function with POx-Palbo would forestall recurrence during CT-179 treatment.
As an initial step in combining CT-179 with POx-Palbo, we analyzed the effect of POx-Palbo on MB OLIG2 expression. For this purpose, we re-analyzed prior scRNA-seq data from tumors progressing on POx-Palbo therapy (GEO accession# GSE188672).
We found that the subset of MB cells previously identified as enriched after chronic  including increased expression of PA2G4-regulated genes and CDK4. As CDK4 is a readily druggable target, we were able to show that CDK4 up-regulation contributed functionally to recurrent tumor growth, as blocking CDK4 enhanced CT-179 efficacy.
CT-179 thus integrated well into multimodal regimens with mutual potentiating effects.
OLIG2 plays a critical role in oligodendrocyte function [51] which is essential for brain development [52], and the safety of inhibiting OLIG2 is an important consideration. SHH-MB tumors constitute the pre-dominant tumor type in young children (<3 years of age) as well as in adults (>17 years of age) [3,53]. The <3 years of age cohort, inparticular is at a stage of significant physiological and neurological development, and humans display prolonged myelination well beyond adolescence [54]. Whether CT-179 produces clinically significant myelin toxicity will need to be evaluated. However, OLIG2 function in oligodendrocytes may be fundamentally different from OLIG2 function in tumor cells, where it modulates the chromatin landscape to activate a unique oncogenic program [55]. CT-179 may, therefore, specifically act on tumor cells without harming normal brain. Consistent with this possibility, scRNA-seq analysis showed no change in the size of the population of myelinating oligodendrocytes, and no depletion of immature oligodendrocytes, which were increased by CT-179 treatment. While additional studies of myelination will be needed, our data suggest that CT-179 will not be toxic to myelinating cells.
In summary, this study shows that inhibition of OLIG2-positive MB tumor cells in combination with RT significantly slows MB progression in vivo. CT-179, a novel, small molecule brain penetrate OLIG2 inhibitor, holds significant promise, particularly for the treatment of SHH-driven MB. CT-179 was recently awarded FDA Rare Paediatric Disease Designation for the treatment of MB in September 2020, paving the way for clinical testing of this effective OLIG2 inhibitor in children with SHH-driven MB.

STAR METHODS
Detailed methods are provided in the online version of this paper and include the following: • KEY RESOURCES TABLE         For panels B--D and F, the p values were determined by two-sided Student's t-test.

MATERIALS AVAILABILITY
All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data availability
All scRNA-seq data have been deposited in the publicly available GEO database, under accession number GSE233519.

FCCS-Analysis of data
= • (4) where, j denotes ATTO488 or Rhodamine B, D is diffusion coefficient, τD is diffusion time, and S is the structure parameter determined in calibration measurements.
Diffusion coefficient of OLIG2-eGFP slowed down by DNA-binding, DDNA-bound, was calculated as follows: DNA-bound = ATTO488 2 4 • OLIG2-eGFP, DNA-bound (5) To assess the interaction between OLIG2-eGFP and OLIG2-Tomato, relative crosscorrelation amplitude (RCA) which is the ratio of the number of bound molecules and the total, free and bound, number of OLIG2-eGFP molecules: To support FCCS data, we also calculated from the autocorrelation curves and average count rates OLIG2-eGFP brightness (as reflected by counts per second per molecule (CPM)) which is reporting on OLIG2 homodimerization: OLIG2-eGFP (7) where, FI denotes average fluorescence intensity, i.e. average count rate and N is the average number of OLIG2-eGFP in observation volume determined from the amplitude of the corresponding autocorrelation curve.

FCCS-Software and statistical analysis
Statistical analysis was performed using 2-sided Student's t-test in Microsoft Excel.
Dose-response curve fitting was performed using OriginPro 2018, Data Analysis and Graphing Software (OriginLab Corporation, USA).

KINOMEscan assay
Effect of CT-179 on protein kinase activity was evaluated using the Thermofisher Scientific SelectScreen Kinase Profiling Service. Over 400 human protein kinases were tested in the presence of 1 μM final concentration of CT-179 at an ATP concentration equal to the known ATP Km for each kinase. The experiment was done by Curtana Pharmaceuticals. Data is presented as percentage of inhibition of enzyme activity. Software CORAL was used to make the kinome tree, which is available from: http://phanstiel-lab.med.unc.edu/CORAL/.

Luciferase reporter assay
Promoter region of human LHX8 was amplified by PCR from human genomic DNA

In vivo pharmacokinetic studies of CT-179 in mice
The in vivo pharmacokinetic studies (EXT-240 and EXT-241) were performed under contract by Biodura Inc.

OLIG2 siRNA knockdown
OLIG2 siRNA#1, #2, #3, and scrambled control oligo sequences were purchased from Sigma-Aldrich. OLIG2 knockdown was done using Lipofectamine TM RNAiMAX transfection reagent with 30 pmol of siRNA. Knockdown efficiency was confirmed by examining OLIG2 expression at mRNA and protein levels.

Irradiation of cells
Cells were seeded into 96-well plates at 10,000 cells/well before irradiation in 137 Cs source gamma rays to achieve 2 Gy (MDS Nordion Gammacell Irradiator).

Cell viability assays
Cell proliferation was determined by a CellTiter 96 ® Non-Radioactive Cell Proliferation Assay (Promega) kit. Absorbance at 490 nm was measured on a Biotek PowerWave (Biotek, USA) plate scanner to obtain raw data. Average absorbance from triplicate wells were plotted against concentration using GraphPad Prism software v.7.0 (GraphPad software). At the indicated time points, cells were stained using FITCconjugated Annexin V and Annexin V binding buffer (BD Pharmingen TM ) according to the manufacturer's instructions. Annexin V or propidium iodide (PI)-positive cells were analyzed using a Fortessa 5 Flow Cytometer (Becton Dickinson) and data was analyzed using FlowJo ® software (Tree Star).

CT-179 drug treatment
The synthesis of CT-179 is described in published patent application WO2016138479A1. To prepare the drug for oral gavage, CT-179 was dissolved in 0.5% (w/v) methylcellulose 4000/0.5% (v/v) Tween 80 in injection water. To prepare the drug for in vitro studies and for intraperitoneal (IP) injection, CT-179 was dissolved in injection water and stored at -80 °C.

Cell cycle analysis
Cell cycle analysis was performed as descried previously [58]. Cells were fixed with 70% ice-cold ethanol and subsequently stained with PI and analyzed using a Fortessa 5 Flow Cytometer (Becton Dickinson) and data was analyzed using FlowJo ® software (Tree Star).
For immunofluorescent staining of mouse MB after CT-179 treatment, brains with tumors were fixed, sectioned, stained and imaged using an Aperio Scanscope as previously described [59].

Immunoblot analysis
Whole cell protein lysate (60 μg/sample) was used. Samples were run on denaturing sodium dodecylsulfate-polyacrylamide gel electrophoresis gels (12%) before transferring onto Immuno-Blot™ polyvinylidene fluoride membranes (Bio-Rad). The reference protein β-actin was used as a loading control.

Data analysis
The scRNA-seq data were analyzed using Parse software to identify cells by bar code, and to map transcripts to the mouse transcriptome. Data were then analyzed using our previously reported pipeline [18,33].

Tissues and organoids
Tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin and, subsequently, stained with haematoxylin and eosin (H&E). IHC on G-Smo mice was as previously described [18]. Antigen retrieval was performed using a pH 9.0 Tris-EDTA buffer. Tissue sections (4 µm) were probed with anti-OLIG2 (Millipore) antibody overnight and subsequently stained with Mach Polymer HRP (Biocare Medical).

GEMM MBs
Brains including tumors from G-Smo mice were harvested, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin at the UNC Centre for Gastrointestinal Biology and Disease Histology core. Sections were deparaffinised, and antigen retrieval was performed using a low-pH citric acid-based buffer. Staining was performed and stained slides were digitally scanned using the Leica Biosystems Aperio ImageScope software (12.